Motility is one of the characteristic features of all living organisms and involves the transduction of chemical into mechanical energy. Nature has evolved a limited number of molecules that accomplish this task. For the linear motors this consists of the myosin, dynein, and kinesin superfamilies of proteins. Understanding how these molecules function at the molecular level continues to be one of the outstanding questions in biology. The primary objective of this proposal is to provide a molecular basis for understanding myosin-based motility by studying the three-dimensional structure of myosin and its interaction with actin. This will be achieved through a combination of single crystal x-ray diffraction and site-directed mutagenesis. These studies will be augmented with kinetic, spectroscopic, and motility measurements on the same proteins. The first set of goals of this proposal is directed toward understanding the conformational changes that occur during the contractile cycle and how they influence the kinetic mechanism of energy transduction by myosin. The focus will be on the molecular events that occur as myosin binds to actin. This will be achieved by trapping the molecule in conformations that resemble its structure toward the end of the power stroke and by determining the structure of the actomyosin complex. In parallel, the role of amino acid residues that have been implicated in enzymatic activity and communication between the nucleotide binding site and the actin interface will be examined. These investigations will be accomplished by studying the motor domain of myosin II from Dictyostelium discoideum. Where appropriate, complementary investigations in Dictyostelium myosin subfragment-1 will be performed. A long-term goal of this proposal is to determine the structure of myosin from other classes of the superfamily. These myosins participate in a wide range of cellular functions and have been implicated in several genetic diseases. They also exhibit very different kinetic and motile properties including processivity and reversal of direction. The purpose of these studies is to understand those factors that influence the kinetic properties of myosin and control its direction of movement on actin.